Our overall goal in this research has been and continues to be to design and develop a genital herpes vaccine(s) that would be effective around the world. Although considerable human disease is caused by herpes simplex viruses, there is no approved vaccine for HSV-1 or HSV-2. We have designed a replication- defective HSV-2 mutant viral strain that is efficacious in two different animal species. In the previous grant period we conducted characterization of our genital herpes vaccine candidate, HSV-2 dl5-29, and this vaccine technology was licensed to Sanofi Pasteur and they are developing and producing it for clinical trials. This was a major step forward for the advancement of this vaccine concept. However, there is a high prevalence of genital herpes in developing countries, in particular Sub-Saharan Africa, and genital herpes is associated with a 3-fold increased risk of HIV infection. We have found that our HSV-2 dl5-29 vaccine candidate induces protection against South African viruses in a murine model, but the protection against the South African viruses is not as good as against US viruses. Furthermore, the South African viruses show increased disease and replication in the animals immunized with all of the vaccines. Therefore, we hypothesize that the South African viruses show antigenic diversity and pathogenetic diversity, which we will explore in this proposed research. Research proposed in this application is aimed at modifying and further improving this vaccine candidate to optimize protection against viruses from Sub-Saharan Africa where there is a co-epidemic of HSV-2 and HIV infection. In this application our specific aims are to: 1. Test the hypothesis that there is antigenic and pathogenetic diversity among HSV-2 strains from around the world and that specific viral genetic backgrounds will need to be used as vaccine strains for optimal efficacy in Sub-Saharan Africa by a) determining the optimal genetic background for a replication-defective HSV-2 strain for use as a vaccine to immunize against HSV-2 strains from Sub-Saharan Africa and b) determining the basis for the difference in the pathogenesis of infection by clinical strains from the US versus South Africa. 2. Test the hypothesis that genetic modifications in immune evasion genes can improve the dl5-29 herpes vaccine candidate by a) replacing the UL41-2 virion host shutoff gene with the homologous HSV-1 gene, UL41-1, containing mutations in the RNase domain and b) by introducing mutations in the US3 gene, whose product inhibits interferon and inflammatory cytokine responses. 3. Test the hypothesis that mucosal priming with an HSV-1 vector expressing HSV-2 glycoproteins B and/or D followed by boosting with HSV-2 dl5-29 will provide superior protection against HSV-2 genital challenge as compared with immunization with dl5-29 virus alone.